Direct cutaneous exposure to vesicants such as nitrogen mustard or nitrogen mustard-related nitrosoureas (BCNU (carmustine)) produce dose-dependent symptoms that are varied in onset, severity and duration of wound healing. Although exposure to the vesicant itself evokes a primary injury (1st hit), the direct insult to the skin results in a delayed influx of activated hyper-inflammatory monocyte/macrophages (M) that constitute an additional insult to the skin (2nd hit) that promotes catastrophic skin injury. However, the interval between the first and second tissue hits represents a countermeasure opportunity. Attenuation of M activation by vitamin D has been reported, and in a clinical anecdotal confirmation of this effect, we have recently treated a patient who exhibited a persistent toxic skin response following topical BCNU treatment using oral vitamin D. This success leads us to hypothesize that attenuating hyper-activated M with 25(OH) D3 will be an effective countermeasure that halts progression of skin destruction, a key morbidity factor following exposure to vesicants. The multidisciplinary translational approach presented here that takes advantage of a strong infrastructure in the Departments of Dermatology and Medicine at Case Western Reserve University and University Hospitals Case Medical Center. Human clinical trial samples as well as murine in vivo and in vitro samples will be used assess the potential for vitamin D to act as a countermeasure to skin damage elicited by M activation following nitrosurea and nitrogen mustard exposure. We have organized a team of national experts to evaluate our progress and help identify complementary approaches to the proposed work. Using human clinical samples obtained from ongoing trials of alkylating agents as well as murine skin samples obtained post-treatment with mechlorethamine we propose to assess: 1.) The level of M infiltration and activation using induction of the soluble signaling molecules TNFa and iNOS in pre- and post-BCNU exposed tissue. We will further 2.) Determine whether vitamin D supplementation modulates tissue destruction and if this is mediated by changes in M activation. Use of a macrophage-restricted VDR knockout mouse system will confirm the specificity of M mediated response. Finally, we will 3.) Begin a vitamin D interventional trial utilizing patients exposed to topical BCNU and nitrogen mustard to assess alteration of inflammatory biomarkers, cellular infiltration and immunohistochemical staining of hyper-activated M in patients while monitoring changes in serum vitamin D levels from baseline to post-therapy. Serum collected pre- and post- supplementation will be assayed for direct suppression of autologous macrophage activation in vitro. Successful implementation of this strategy is likely to save lives in the event of mass exposure as vitamin D is safe and widely available. Demonstration that vitamin D-related compounds protect against tissue destruction mediated by vesicants will lead to advanced evaluation of this potential therapeutic intervention. PUBLIC HEALTH RELEVANCE: Chemicals vesicants such as nitrogen mustard or nitrogen mustard-related nitrosureas (BCNU) used as weapons can have a devastating impact on human health. The mustards are powerful alkylating agents and exposure to these agents by epithelial cells, such as keratinocytes, result in induction of oxidative and nitrosative stress an eventual cell death. In the skin, this cell death manifests as severe skin blistering and desquamation. New therapeutics which can halt the effects of these chemicals will be invaluable to treating these skin injuries. Using both mouse and human tissue this proposal addresses whether vitamin D can be used as a counteragent to the activation and subsequent tissue destruction caused hyper-activated macrophages initiated through vesicant exposure.